Anti mouse f4 80 antibody

SnMP, DNR hydrochloride, poly(lactic‐co‐glycolic acid) (PLGA, lactide: glycolide 50:50, 7000–17000 Da), and biotin‐FITC were purchased from Sigma Aldrich (St. Louis, MO, USA). 1,2‐Dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) and 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[biotinyl(polyethylene glycol)‐2000] (DSPE‐PEG₂₀₀₀‐Biotin), 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[amino(polyethylene glycol)‐2000] (DSPE‐PEG₂₀₀₀) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Anti‐human CD33, CD64 antibodies and anti‐mouse CD11b, CD45, CD206, Ly6c, Gr1, TNF‐α, IL12p70, Rat IgG1 Isotype antibodies were purchased from BD Biosciences (USA) (Table 1, Supporting Information). Anti‐mouse F4/80 antibody and Avidin‐FITC were purchased from Biolegend (San Diego, CA, USA). Anti‐human CCR2 antibody was obtained from R&D Systems (Minneapolis, MN, USA). Anti‐His‐Tag, anti‐human HO1 (P249), and β‐actin antibodies (13E5) were obtained from Cell Signaling Technology (Danvers MA, USA).

Glrx knockout (KO) mice were generated by Dr. Y-S. Ho (Wayne State University, MI) [28 (link)]. Homozygous Glrx KO and genetically-matched wild type (WT) control mice were transferred and bred in the Laboratory Animal Science Center (LASC) on the Boston University Medical Campus as approved by Institutional Animal Care and Use Committee at Boston University. Mice were euthanized by carbon dioxide inhalation according to a protocol approved by Boston University Animal Care and Use Committee. Peritoneal macrophages were collected from WT and KO mice (8–10 months old) by injection of cold PBS containing glucose into the peritoneal cavity immediately after euthanasia and collection of peritoneal fluid. Cells from 4–5 mice were pooled and centrifuged at 1000 rpm, re-suspended in DMEM containing 10% FBS, and plated into 24 wells. After one hour, cells were washed with warm PBS to remove non-adherent cells and adherent cells were cultured in DMEM +10% FBS with penicillin-streptomycin [29 (link)]. The following day the medium was changed, and cells were treated with LPS (100 ng/ml) for 6 hours followed by RNA extraction. Before LPS stimulation an aliquot of cells was fixed with 4% paraformaldehyde and stained with anti-mouse F4/80 antibody (BioLegend). F4/80 positive cells assessed by Image J of total cell number which was counted by Hoechst staining,

Pyrrolidine dithiocarbamate (PDTC) and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nigericin was purchased from Tocris (Bristol, UK). Salmonella typhimurium flagellin and poly (dA:dT) were purchased from InvivoGen (San Diego, CA, USA). Anti-phospho-IκB, anti-phospho-NF-κB (p65), anti-IL-6, and anti-COX-2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-IL-1β antibody was purchased from R&D Systems (Minneapolis, MN, USA). Anti-NLRP3 and anti-caspase-1 antibodies were purchased from AdipoGen Life Science (San Diego, CA, USA). The anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-mouse F4/80 antibody was purchased from Biolegend (San Diego, CA, USA).

Non‐parenchymal cells (NPCs) were isolated from whole livers (sham) or ischaemic lobes (I/R) following 12 hours of reperfusion. Cells were stained with antimouse F4/80 antibody (BioLegend), Ly6G (BioLegend) and CD11b (BioLegend) to detect neutrophils and macrophages infiltration according to the manufacturer's protocol. We defined Kupffer cells as CD11b⁺F4/80⁺ and neutrophils as CD11b⁺Ly6G⁺. After 30 minutes incubation at 37°C in the dark, suspensions were washed with PBS and suspended with 300 μL PBS. Samples were determined and sorted by FAC‐SCanto^™ II flow cytometer (BD Biosciences), and the data were analysed with Flow Jo (Tree Star).